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Malaysian Journal of Medical Sciences
School of Medical Sciences, Universiti Sains Malaysia
ISSN: 1394-195X
Vol. 20, No. 5, 2013, pp. 16-22
Bioline Code: mj13067
Full paper language: English
Document type: Research Article
Document available free of charge

Malaysian Journal of Medical Sciences, Vol. 20, No. 5, 2013, pp. 16-22

 en Purification of an IgA Monoclonal Antibody Specific for the Acr Protein of Mycobacterium tuberculosis check for this species in other resources by Immunoaffinity Chromatography
Reyes, Fátima; Otero, Oscar; Camacho, Frank; Sarmiento, María Elena & Acosta, Armando


Background: A monoclonal antibody (mAb) of the IgA isotype, designated TBA61, is specific for the Acr protein of Mycobacterium tuberculosis check for this species in other resources (MTB). TBA61 has been used in studies exploring protection against tuberculosis (TB), and its efficacy has been proven using different challenge models. To purify the mouse IgA isotype, a combination of methods, such as globulin precipitation, ion exchange, and gel filtration, is usually required to achieve a satisfactory degree of purity.
Methods: To minimise the number of chromatographic steps, we proposed to employ immunoaffinity chromatography using the Acr protein of MTB as a specific ligand for this mAb. For this purpose, the HspX gene was cloned and expressed in Escherichia coli check for this species in other resources , and recombinant Acr (rAcr) was coupled to a cyanogen bromide-activated Sepharose 4B matrix, which was used to purify TBA61 mAb from ascites produced in mice in a single step.
Results: The recovery from the purification procedure was 1.46 mg per mL of ascites. Analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot showed a high purity. The purified mAb retained its reactivity against the Acr protein based on enzyme-linked immunosorbent assay (ELISA) and western blot.
Conclusion: The purification method used is rapid, simple, and specific and can be easily scaled up.

Mycobacterium tuberculosis; IgA; monoclonal antibody; Acr protein; affinity; chromatography

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